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A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling.

Kirsch K, Kensinger M, Hanafusa H, August A - BMC Cell Biol. (2002)

Bottom Line: A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling.We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation.This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Oncology, The Rockefeller University, NY, NY 10021, USA. kirschk@bu.edu

ABSTRACT

Background: The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.

Results: We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

Conclusion: Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

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Tyrosine phosphorylated Cas substrate domain affects c-crk interaction with C3G, but not endogenous FAK or Cas tyrosine phosphorylation.a) Lysates from NIH3T3 cells stably carrying vector alone or expressing the Src*/Cas(SD) chimera were immunoprecipitated with anti-phosphotyrosine (top panel), or anti-crk (middle and bottom panels) and probed with anti-HA to detect the Src*/Cas(SD) chimera (top and middle panels), or C3G (bottom panel). Lanes 1 and 2, vector and the Src*/Cas(SD) chimera transfected NIH3T3 cells respectively, whole cell extract; lanes 3 and 4, vector transfected NIH3T3 cells immunoprecipitated with anti-phosphotyrosine antibody; lane 5, NIH3T3 cells expressing the Src*/Cas(SD) chimera immunoprecipitated with anti-phosphotyrosine antibody. Arrows indicate the Src*/Cas(SD) chimera and C3G in the top, middle and bottom panels. b) A duplicate blot to that shown in (a) was probed with anti-Fak antibodies (top panel) or with anti-Cas antibodies (bottom panel). Arrows indicate FAK and Cas.
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Figure 7: Tyrosine phosphorylated Cas substrate domain affects c-crk interaction with C3G, but not endogenous FAK or Cas tyrosine phosphorylation.a) Lysates from NIH3T3 cells stably carrying vector alone or expressing the Src*/Cas(SD) chimera were immunoprecipitated with anti-phosphotyrosine (top panel), or anti-crk (middle and bottom panels) and probed with anti-HA to detect the Src*/Cas(SD) chimera (top and middle panels), or C3G (bottom panel). Lanes 1 and 2, vector and the Src*/Cas(SD) chimera transfected NIH3T3 cells respectively, whole cell extract; lanes 3 and 4, vector transfected NIH3T3 cells immunoprecipitated with anti-phosphotyrosine antibody; lane 5, NIH3T3 cells expressing the Src*/Cas(SD) chimera immunoprecipitated with anti-phosphotyrosine antibody. Arrows indicate the Src*/Cas(SD) chimera and C3G in the top, middle and bottom panels. b) A duplicate blot to that shown in (a) was probed with anti-Fak antibodies (top panel) or with anti-Cas antibodies (bottom panel). Arrows indicate FAK and Cas.

Mentions: We then used the 5' and 3' fusions of the Cas SD to determine which motif, YQXP or YDXP, interacts with the v-crk SH2 domain. Expression of the SrcKM fused to the full length Cas SD, 5' N-terminal YQXP motifs or 3' C-terminal YDXP motifs alone demonstrated that these fusions were not stably tyrosine phosphorylated in cells (Fig. 4b, lanes 1–3), although they can be stably tyrosine phosphorylated when fused to the attenuated Src kinase domain (data not shown and see Fig. 7a). By contrast, co-expression of these fusions along with v-crk results in stable tyrosine phosphorylation of the 3' C-terminal YDXP motifs and the full length Cas SD (carrying both motifs), but not the 5' N-terminal YQXP motifs (Fig. 4b, lanes 4–6). These data indicate that the v-crk SH2 domain interacts with the C-terminal YDXP motifs, and as suggested by Birge et al, v-crk may be able to protect them from dephosphorylation and/or induces their phosphorylation [12]. They also suggest that the YQXP motifs may be involved in binding to other intracellular signaling molecules.


A p130Cas tyrosine phosphorylated substrate domain decoy disrupts v-crk signaling.

Kirsch K, Kensinger M, Hanafusa H, August A - BMC Cell Biol. (2002)

Tyrosine phosphorylated Cas substrate domain affects c-crk interaction with C3G, but not endogenous FAK or Cas tyrosine phosphorylation.a) Lysates from NIH3T3 cells stably carrying vector alone or expressing the Src*/Cas(SD) chimera were immunoprecipitated with anti-phosphotyrosine (top panel), or anti-crk (middle and bottom panels) and probed with anti-HA to detect the Src*/Cas(SD) chimera (top and middle panels), or C3G (bottom panel). Lanes 1 and 2, vector and the Src*/Cas(SD) chimera transfected NIH3T3 cells respectively, whole cell extract; lanes 3 and 4, vector transfected NIH3T3 cells immunoprecipitated with anti-phosphotyrosine antibody; lane 5, NIH3T3 cells expressing the Src*/Cas(SD) chimera immunoprecipitated with anti-phosphotyrosine antibody. Arrows indicate the Src*/Cas(SD) chimera and C3G in the top, middle and bottom panels. b) A duplicate blot to that shown in (a) was probed with anti-Fak antibodies (top panel) or with anti-Cas antibodies (bottom panel). Arrows indicate FAK and Cas.
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Figure 7: Tyrosine phosphorylated Cas substrate domain affects c-crk interaction with C3G, but not endogenous FAK or Cas tyrosine phosphorylation.a) Lysates from NIH3T3 cells stably carrying vector alone or expressing the Src*/Cas(SD) chimera were immunoprecipitated with anti-phosphotyrosine (top panel), or anti-crk (middle and bottom panels) and probed with anti-HA to detect the Src*/Cas(SD) chimera (top and middle panels), or C3G (bottom panel). Lanes 1 and 2, vector and the Src*/Cas(SD) chimera transfected NIH3T3 cells respectively, whole cell extract; lanes 3 and 4, vector transfected NIH3T3 cells immunoprecipitated with anti-phosphotyrosine antibody; lane 5, NIH3T3 cells expressing the Src*/Cas(SD) chimera immunoprecipitated with anti-phosphotyrosine antibody. Arrows indicate the Src*/Cas(SD) chimera and C3G in the top, middle and bottom panels. b) A duplicate blot to that shown in (a) was probed with anti-Fak antibodies (top panel) or with anti-Cas antibodies (bottom panel). Arrows indicate FAK and Cas.
Mentions: We then used the 5' and 3' fusions of the Cas SD to determine which motif, YQXP or YDXP, interacts with the v-crk SH2 domain. Expression of the SrcKM fused to the full length Cas SD, 5' N-terminal YQXP motifs or 3' C-terminal YDXP motifs alone demonstrated that these fusions were not stably tyrosine phosphorylated in cells (Fig. 4b, lanes 1–3), although they can be stably tyrosine phosphorylated when fused to the attenuated Src kinase domain (data not shown and see Fig. 7a). By contrast, co-expression of these fusions along with v-crk results in stable tyrosine phosphorylation of the 3' C-terminal YDXP motifs and the full length Cas SD (carrying both motifs), but not the 5' N-terminal YQXP motifs (Fig. 4b, lanes 4–6). These data indicate that the v-crk SH2 domain interacts with the C-terminal YDXP motifs, and as suggested by Birge et al, v-crk may be able to protect them from dephosphorylation and/or induces their phosphorylation [12]. They also suggest that the YQXP motifs may be involved in binding to other intracellular signaling molecules.

Bottom Line: A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling.We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation.This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Molecular Oncology, The Rockefeller University, NY, NY 10021, USA. kirschk@bu.edu

ABSTRACT

Background: The adaptor protein p130Cas (Cas) has been shown to be involved in different cellular processes including cell adhesion, migration and transformation. This protein has a substrate domain with up to 15 tyrosines that are potential kinase substrates, able to serve as docking sites for proteins with SH2 or PTB domains. Cas interacts with focal adhesion plaques and is phosphorylated by the tyrosine kinases FAK and Src. A number of effector molecules have been shown to interact with Cas and play a role in its function, including c-crk and v-crk, two adaptor proteins involved in intracellular signaling. Cas function is dependent on tyrosine phosphorylation of its substrate domain, suggesting that tyrosine phosphorylation of Cas in part regulates its control of adhesion and migration. To determine whether the substrate domain alone when tyrosine phosphorylated could signal, we have constructed a chimeric Cas molecule that is phosphorylated independently of upstream signals.

Results: We found that a tyrosine phosphorylated Cas substrate domain acts as a dominant negative mutant by blocking Cas-mediated signaling events, including JNK activation by the oncogene v-crk in transient and stable lines and v-crk transformation. This block was the result of competition for binding partners as the chimera competed for binding to endogenous c-crk and exogenously expressed v-crk.

Conclusion: Our approach suggests a novel method to study adaptor proteins that require phosphorylation, and indicates that mere tyrosine phosphorylation of the substrate domain of Cas is not sufficient for its function.

Show MeSH
Related in: MedlinePlus